Viral vector compositions and methods of use thereof
AssigneeLogicBio Therapeutics, Inc.
InventorsQiang Xiong, B. Nelson Chau
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Abstract
Presented herein are compositions and methods for improved gene editing with AAV vector constructs. Among other things, the present disclosure recognizes that homology arms of a certain length (e.g., at least 750 nt or at least 1000 nt each) may demonstrate improved editing activity. In some embodiments, the present disclosure recognizes that homology arms of a certain length (e.g., at least 750 nt or at least 1000 nt each) may demonstrate additional improvements in editing activity when homology arms are of different lengths.
Claims29
A recombinant viral vector for integrating a transgene into a target integration site in the genome of a cell, comprising:
- (i) a polynucleotide comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises at least one exogenous gene sequence and the second nucleic acid sequence is positioned 5′ or 3′ to the first nucleic acid sequence and promotes the production of two independent gene products upon integration into a target integration site;
- (ii) a third nucleic acid sequence positioned 5′ to the polynucleotide and comprising a sequence that is homologous to a genomic sequence 5′ of the target integration site;
- and
- (iii) a fourth nucleic acid sequence positioned 3′ to the polynucleotide and comprising a sequence that is homologous to a genomic sequence 3′ of the target integration site;
- wherein each of the third nucleic acid sequence and fourth nucleic acid sequence are at least 1000 nucleotides (nt) in length and wherein the third nucleic acid sequence and fourth nucleic acid sequence are different lengths.
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A method of determining homology arm lengths for a polynucleotide cassette comprising at least one payload, a 5′ homology arm, and a 3′ homology arm comprising
- (a) determining the length of a polynucleotide cassette comprising at least one payload but not including any homology arms,
- (b) if the lengths of step (a) is less than 2.7 kilobases (kb), then the length of the 3′ homology arm sequence is 1 kb or less and the length of the 5′ homology arm sequence is 2.7 kb—the length of the 3′ homology arm, and
- (c) if the length of step (a) is greater than 2.7 kb, then the 5′ and 3′ homology arms are each [4.7 kb—the length of step (a)]/2 nucleotides in length
Dependencies are inferred from the claim text.